Design, synthesis and biological activity of N5-substituted tetrahydropteroate analogs as non-classical antifolates against cobalamin-dependent methionine synthase and potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2020.112113

Cobalamin-dependent methionine synthase (MetH) is involved in the process of tumor cell growth and survival. In this study, a novel series of N⁵-electrophilic substituted tetrahydropteroate analogs without glutamate residue were designed as non-classical antifolates and evaluated for their inhibitory activities against MetH. In addition, the cytotoxicity of target compounds was evaluated in human tumor cell lines. With N⁵-chloracetyl as the optimum group, further structure research on the benzene substituent and on the 2,4-diamino group was also performed. Compound 6c, with IC₅₀ value of 12.1 μM against MetH and 0.16–6.12 μM against five cancer cells, acted as competitive inhibitor of MetH. Flow cytometry studies indicated that compound 6c arrested HL-60 cells in the G₁-phase and then inducted late apoptosis. The molecular docking further explained the structure-activity relationship.

Full text of DOI:10.1016/j.ejmech.2020.112113

Journal Pre-proof
5
Design, synthesis and biological activity of N -Substituted tetrahydropteroate analogs
as non-classical antifolates against cobalamin-dependent methionine synthase and
potential anticancer agents
Meng Wang, Chao Tian, Liangmin Xue, Hao Li, Jing Cong, Fang Fang, Jiajia Yang,
Mengmeng Yuan, Ying Chen, Ying Guo, Xiaowei Wang, Junyi Liu, Zhili Zhang
PII:
S0223-5234(20)30080-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112113
EJMECH 112113
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 December 2019
Revised Date: 28 January 2020
Accepted Date: 31 January 2020
Please cite this article as: M. Wang, C. Tian, L. Xue, H. Li, J. Cong, F. Fang, J. Yang, M. Yuan, Y.
5
Chen, Y. Guo, X. Wang, J. Liu, Z. Zhang, Design, synthesis and biological activity of N -Substituted
tetrahydropteroate analogs as non-classical antifolates against cobalamin-dependent methionine
synthase and potential anticancer agents, European Journal of Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.ejmech.2020.112113.
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2020 Published by Elsevier Masson SAS.

5
Design, Synthesis and Biological Activity of N -Substituted
Tetrahydropteroate Analogs as Non-classical Antifolates against
Cobalamin-dependent Methionine Synthase and Potential Anticancer
Agents
*
,†,§
*,†
†
†
†
†
†
Meng Wang,
Chao Tian, Liangmin Xue, Hao Li, Jing Cong, Fang Fang, Jiajia Yang, Mengmeng
†
†
†
†
†, ‡,
⊥
†,
⊥
Yuan, Ying Chen, Ying Guo, Xiaowei Wang, Junyi Liu,
Zhili Zhang
†
Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
College of Pharmacy, Beihua University, Jilin 132013, China
‡
§
ABSTRACT: Cobalamin-dependent methionine synthase (MetH) is involved in the process of tumor cell
5
growth and survival. In this study, a novel series of N -electrophilic substituted tetrahydropteroate
analogues without glutamate residue were designed as non-classical antifolates and evaluated for their
inhibitory activities against MetH. In addition, the cytotoxicity of target compounds was evaluated in
5
human tumor cell lines. With N -chloracetyl as the optimum group, further structure research on the
benzene substituent and on the 2,4-diamino group was also performed. Compound 6c, with IC50 value of
1
2.1 µM against MetH and 0.16-6.12 µM against five cancer cells, acted as competitive inhibitor of MetH.
Flow cytometry studies indicated that compound 6c arrested HL-60 cells in the G -phase and then inducted
1
late apoptosis. The molecular docking further explained the structure-activity relationship.
Keywords: pyrido[3,2-d]pyrimidine, methionine synthase, antifolate, inhibitor, anticancer
1
. INTRODUCTION
Cobalamin-dependent methionine synthase (MetH), an important regulator of folate metabolism [1],
catalyzes the transfer of methyl groups from the cofactor methyltetrahydrofolate (MTHF) to homocysteine
Hcy) to produce tetrahydrofolate (THF) and methionine (Met) [2].The cobalamin cofactor of MetH plays a
(
crucial role in catalytic reaction which circulates between +1 and +3 oxidation states as methyl acceptor
and methyl donor. During MetH reaction cycle the transfer of methyl group from MTHF to Met is in two
steps by two nucleophilic substitution reactions. Firstly, the methyl group is transferred from MTHF to
cofactor cob(I)alamin (Cbl(I)) to generate methylcob(III)alamin. Secondly, the methylcob(III)alamin is
demethylated by Hcy to form cob(I)alamin again (Figure 1) [3-5].
⊥
Corresponding author. Tel.: +86 10 82805203.
E-mail address: lilybmu@bjmu.edu.cn (J. Liu).
Chao Tian (co-first author)

Figure 1. The cobalamin-dependent methionine synthase reaction.
Both cobalamin-dependent and cobalamin-independent forms of methionine synthase could be found in
microorganisms, while the former in mammalian tissues and the latter in higher plant [6-8]. In humans, the
primary circulating folate is MTHF, with sufficient physiological concentrations of ~5-30 nM in the blood
[9]. It is well known that MetH is the only human enzyme that metabolizes MTHF to regenerate the active
form THF. Therefore, it is essential in many important biochemical pathways, including folate cycles and
one-carbon methionine transmethylation [10, 11]. In folate cycles, THF is transformed to other bioactive
folates that provide one-carbon units for purine and pyrimidine synthesis [12, 13]. Met, a nutritionally
essential amino acid and component of proteins, can be transformed to the active s-adenosyl methionine
(SAM) which is an important methyl donor involved in biologically methylations of DNA, RNA, proteins,
lipids and polyamine [14, 15]. Thus, impaired function of MetH could provide a valuable target for
chemotherapeutic intervention in cancer [16].
Although it is recognized that MetH is an excellent target for rational drug design, there are a few studies
on MetH inhibitors for anticancer. It was reported that some non-drug like inhibitors, including nitrous
oxide (N O) [17], nitric oxide (NO) [18, 19], sodium nitroprusside [20] and ethanol [21], inhibited the
2
enzyme through oxidation of the cobalamin cofactor. Methylmercury [22] and cobalamin analogs [23-25]
were reported as non-drug like inhibitors too. However, there is less study on drug-like MetH inhibitors.
2

Figure 2. Structures of recently published MetH inhibitors.
Recently, novel benzothiadiazole (1), benzimidazole (2), quinoxaline (3) and analogues of quinazoline
derivatives (4) were designed to mimic the substructure of MTHF, and evaluated for their activities against
5
purified rat liver MetH (Figure 2) [26-28]. N -substituted 8-deazatetrahydrofolates (5) and aziridine
tetrahydrofolate analogs (6) were designed and synthesized in our previous work, which could bind to
MetH in place of MTHF by the formation of covalent bonds with the nucleophilic Cbl(I) cofactor and then
inhibit methyl transfer [29, 30]. Though exhibiting certain inhibitory activities against MetH of HL-60 cells,
these compounds share the shortcomings of classical antifolates because of their folate-like structures
including the glutamic acid side chain. Classical antifolates, such as methotrexate (MTX), raltitrexed
(RTX), pemetrexed (PMX) and pralatrexate (Figure 3) can’t passively diffuse across cell membranes and
must be actively transported using folate receptors (FRα, FRβ, FRγ), reduced folate carrier (RFC), or the
5
proton-coupled folate transporters (PCFT) [31-34]. And, N -substituted 8-deazatetrahydrofolates and
aziridine tetrahydrofolate analogs [29, 30] undergo the same intracellular metabolic processing, where
folylpolyglutamate synthase (FPGS)-mediated polyglutamylation is a crucial determinant of their
intracellular retention and pharmacological activity [35]. The impairment of the destroyed transport and
polyglutamation mechanisms through genetic mutation can cause the resistance, which is the obvious
shortcoming of classical anfitolates [36].
3

Figure 3. Structures of classical and nonclassical antifolates.
In contrast, nonclassical antifolates without glutamic acid moiety can penetrate cells through passive
diffusion and act with no need of polyglutamylation, thus can overcome resistance of classical antifolates.
Lipophilic nonclassical antifolates [37, 38], such as piritrexim and trimetrexate (Figure 3), have
demonstrated significant growth inhibition of tumor cell. There has been a continuing effort in our
5
laboratory to develop novel nonclassical MetH inhibitor based on N -position substituted
8
-deazatetrahydrofolates. The aim of the present study was to design and synthesize more lipophilic
derivatives that lack a glutamate tail and to explore structure-activity relationships within a series of
5
N -position substituted tetrahydropyrido[3,2-d]pyrimidines and different substituents of benzene ring
(Figure 4).
5
Figure 4. Design of novel N -substituted tetrahydropyrido[3,2-d]pyrimidines.
4

2
. RESULTS AND DISCUSSION
5
2
.1 Design and synthesis of N -substituted compounds in series 1.
5
Among the N -substituted tetrahydropteroate analogues, compounds with dibromopropyl and formyl
5
groups on N position showed the greatest activities against MetH with IC of 1.43-8.11 µM (Figure 4)
5
0
[29]. So, more electrophilic groups, such as formyl, acryloyl, isopropanecarbonyl, chloroacetyl, chloroethyl,
5
3
-chloropropionyl, 3-chloropropyl and bromoacetyl groups, were designed on N -position to research the
influence of electrophilicity, size and steric hindrance of substituents besides truncation of the glutamate
tail present in classical antifolates. Considering the potential flexibility and better lipid solubility, a C−C
bridge between pyridopyrimidine and aryl ring is necessary compared with C-N bridge [39-42], thus
leading to our first design of the compound series 1 (Figure 4).
o
Scheme 1. Reagents and conditions: (a) p-toluenesulfonamide, N,N-dimethylacetamide, 160 C, 36 h; (b)
o
POCl , Et N, reflux, 8 h; (c) saturated NH in dry methanol, 150 C, 8 h; (d) Pd/C, ethanol, rt, 12 h; (e)
3
3
3
PtO , ethanol, 0.5 mol/L HCl, rt, 12 h; (f) PtO , ethanol, 2 mol/L HCl, rt, 12 h; (g) 6a : HCOOH,
2
2
1
(
CH CO) O, CH Cl ; 6a , 6a , 6a , 6a , 6a , 6a ,: acryloyl chloride, allyl bromide, isopropanecarbonyl
3 2 2 2 2 3 4 5 7 9
chloride, chloroacetyl chloride, 3-chloropropionyl chloride, bromoacetyl chloride, respectively, anhydrous
DMF, rt; (h) BH -THF, THF, rt.
3
Target compounds 6a -6a were prepared via the synthetic route outlined in Scheme 1. Using the Skraup
1
9
reaction previously described, 5-aminouracil and croton aldehyde were cyclized in 20% HCl to give
2
,4-dihydroxy-6-methylpyrido[3,2-d]pyrimidine (1) in good yield [43]. Then alkenylation of compound 1
5

3
with p-methyl benzaldehyde and p-toluenesulfonamide via sp C-H bond activation in DMAC achieved
(E)-6-(4-methylstyryl)pyrido[3,2-d]pyrimidine-2,4(1H,3H)-dione (2) in one-step in 91% yield [44].
Treatment of compound 2 with a large excess of POCl produced 2,4-dichloride derivative (3) in 65% yield.
3
Then compound 3 was reacted directly with a saturated solution of ammonia in methanol in a sealed vessel
at 150 °C for 8 h to give a light yellow solid (4a) in 95% yield [45]. Hydrogenation of the carbon-carbon
double bonds and pyridine ring in compounds 4a with PtO and 0.5 mol/L HCl in ethanol at room
2
temperature gave compound 5a in excellent yields. When 2 mol/L HCl was used in reaction, cyclohexyl
product 4a-2 was produced. But when Pd/C was used as catalyst, only the double bond was reduced to give
4
a-1. With the reduction product 5a in hand, treatment with a mixture of acetic anhydride and formic acid
5
5
gave the N -formyl derivative 6a directly. The N -chloracetyl and bromoacetyl derivatives 6a , 6a were
1
5
9
obtained in good yields by chloroacetylation and bromoacetylation of 5a. Similarly, compound 5a was
5
5
acylated by 3-chloropropionyl chloride to give N -3-chloropropionyl derivative 6a . The N -chlorinated
7
5
acyl derivatives 6a and 6a were then converted to N -2-chloroethyl derivative 6a and 3-chloropropyl
5
7
6
5
derivative 6a by BH -THF in THF. Other N -acyl and allyl derivatives 6a -6a were obtained by acylation
8
3
2
4
and allylation of 5a in good yields.
.2 Preliminary Evaluation of Compounds 6a -6a .
2
1
9
Table 1 Chemical structures and in vitro inhibitory activities against tumor cells and MetH of the
5
N -position substituted pyrido[3,2-d]pyrimidines in series 1.
b
Tumor cells (IC , µM)
MetH (IC50,
50
1
Compounds
R
b
H1299
>100
A549
HL-60
HeLa
>100
HT29
>100
µM)
c
6
6
6
a1
a2
a3
CHO
74.6±1.1
24.7±0.6
10.8%
c
COCH=CH
18.5±1.3
59.9±1.8
9.93±0.24
1.65±0.24
27.1±4.1
12.9±0.1
46.3±0.8
26.2±0.6
13.4%
2
c
CH
CH=CH
9.75±0.37
9.52±0.31
41.6%
2
2
c
6
a4
>100
>100
12.2±1.1
65.8±4.4
72.5±2.7
22.0%
6
6
a5
a6
COCH
2
Cl
Cl
0.21±0.02
17.7±1.6
1.97±0.03
18.1±0.6
0.39±0.06
2.38±0.05
1.46±0.07 4.74±0.43
21.4±0.9 20.7±0.6
5.66±0.49
c
CH CH
37.5%
2
2
6

c
c
6
6
6
a7
a8
a9
COCH
2
CH
2
Cl
Cl
85.3±5.5
15.9±2.8
0.99±0.19
>100
21.8±1.5
1.04±0.10
1.60±0.16
69.5±6.3
22.8±1.2
>100
20.7±1.8
6.14±0.84
>100
46.9%
CH CH
CH
25.1±2.6
30.0%
2
2
2
a
a
COCH
/
Br
ND
ND
48.0±2.9
2
a
MTX
0.072±0.001 0.014±0.002 0.023±0.001
>100
ND
a
b
c
Not tested. Indicates the average value of three independent experiments. Inhibition% at 100 µM.
5
The N -electrophilic substituted compounds then were tested for their antiproliferative activities against
MetH and five human tumor cell lines. Besides HL-60 (human promyelocytic leukemia), A549 (human
lung) and H1299 (human lung) based on the clinical effect of antifolates MTX and Pemetrexed, HeLa
(cervix) and HT29 (colon cancer) were also chosen because of their relative high incidence [46] (Table 1).
Among the five cell lines, HL-60 cell lines were most sensitive to the cytotoxic effects of target compounds
with the IC s of 0.39-25 µM. While big difference of H1299, A549, HeLa and HT29 cell inhibition
5
0
5
between 6a -6a was observed. 6a and 6a , with the N -chloracetyl and bromoacetyl group respectively,
1
9
5
9
exhibited the notable activities against the tumor cells as IC s of 0.2-4.7 µM and 1.0-6.1 µM. The result is
5
0
consistent with what had been observed in the inhibition against MetH, in which IC values of 6a and 6a
9
5
0
5
(
IC values 5.66 µM and 48.0 µM respectively) were more potent than others (IC values >100 µM).
5
0
5
0
5
It was apparent that the chloracetyl and bromoacetyl groups at N -position were associated with stronger
inhibition against MetH than other subtitutents. They showed excellent activities compared to the MetH
inhibitors reported in literatures such as benzothiadiazoles with IC >95 µM [26], benzimidazole with
5
0
IC >50 µM besides two derivatives with IC value of about 18 µM ,quinoxalines with IC50 >70 µM
50
50
besides only one derivative with IC value of 9 µM and quinazolines with inhibition of 60% at 100 µM [27,
5
0
5
2
8].The significant difference of biochemical activities between 6a (N -chloroacetyl group, 5.66 µM), and
5
5
5
6
a (N -chloroethyl group, >100 µM) and 6a (N -chloropropionyl group, >100 µM) illustrated the
6 7
5
necessity of the carbonyl and β-chloride groups in N substituent. Compound 6a with 5-chloracetyl group
5
were approximately 10-fold more potent than 5-bromoacetyl compound 6a . The corresponding
9
5
5
N -chloroethyl compound 6a was less potent than N -chloracetyl compound 6a whether against tumor
6
5
cells (9.0-87.3 µM verse 0.2-4.7 µM) or MetH (>100 µM verse 5.66 µM).
The results of compounds 6a , 6a , 6a , 6a and 6a indicated that both carbonyl and halogen atom at its
1
5
6
7
9
5
β-position on N -position were necessary for activities against MetH. The function of carbonyl group will
be studied further in the molecular modeling section to understand the binding activity to active site of
7

MetH.
.3 Structure-Activity Relation Research Based on Lead Compound 6a5.
2
o
Scheme 2. Reagents and conditions: (a) p-toluenesulfonamide, N,N-dimethylacetamide, 160 C, 36 h; (b)
o
POCl , Et N, reflux, 8 h; (c) saturated NH in dry methanol, 150 C, 8 h; (d) PtO , 0.5 mol/L HCl, ethanol,
3
3
3
2
rt; (e) acetyl chloride, anhydrous DMF, rt.
5
In view of the structural novelty with N - electrophilic groups, we selected compound 6a as lead
5
inhibitor for further structural optimization. To identify the structure activity relationships of substituents on
benzene, electron-donating groups (ethyl, isopropyl and t-butyl) were chosen as the p-substitution based on
the methyl group of 6a Methoxyl group was also selectived as electron-donation substitutent, and attached
5
.
at different positions on the benzene ring as o-, m- and p-position. Beyond that, halogen
electron-withdrawing groups such as fluorine and chloride were attached at p-position of benzene ring too.
Compounds 6b-6j of series 2 were designed and successfully synthesized (Scheme 2). The target
compounds were tested for their inhibitory activity against MetH of HL-60, and the half-inhibitory
concentrations (IC s) are presented in Table 2.
5
0
Table 2 Chemical structures and in vitro inhibitory activities against tumor cells and MetH of the
5
N -chloroacetyl pyrido[3,2-d]pyrimidines in series 2
a
Tumor cells (IC , µM)
MetH
50
2
Compounds
R
a
H1299
A549
HL-60
HeLa
HT29
(IC , µM)
50
b
6
b
H
0.44±0.06
4.42±0.45
0.63±0.05
1.82±0.60
3.43±0.54
45.8%
8

’
6
c
4 -C
2
H
5
0.16±0.01
0.093±0.018
0.049±0.007
0.23±0.05
0.12±0.02
0.57±0.16
0.24±0.07
0.66±0.02
6.12±0.38
3.13±0.30
3.13±0.30
1.16±0.04
4.35±0.40
6.16±0.33
1.51±0.13
0.53±0.01
0.32±0.01
0.20±0.01
0.79±0.03
0.46±0.01
0.63±0.05
0.34±0.01
0.95±0.05
0.85±0.16
2.16±0.79
0.44±0.16
0.96±0.20
1.52±0.51
2.71±0.12
1.64±0.06
5.25±0.78
3.02±0.18
3.43±0.54
1.14±0.03
2.96±0.27
3.49±0.02
5.62±0.69
5.55±0.52
4.83±0.06
12.1±1.0
45.9±3.6
13.5±1.8
4.99±0.85
14.9±0.78
’
6
d
4 -CH(CH )
3 2
’
6
e
4 -C(CH
3
)
3
’
6
f
4 -F
’
6
6
g
4 -Cl
’
b
h
4 -OCH
3
46.1%
’
b
6
i
3 -OCH
3
42.6%
’
6
j
2 -OCH
3
2.31±0.06
51.5±4.5
a
b
Indicates the average value of three independent experiments. Inhibition% at 100 µM.
5
Compared with the analogues of series 1 (IC =0.21- >100 µM) in Table 1, 6b-6j, with N -chloracetyl
5
0
substituent, exhibited more potent inhibitory activities against all five tumor cells (IC =0.049-6.16 µM).
5
0
5
Besides 6b
, 6h and 6i, all N -chloracetyl compounds (Table 2) showed better activities against MetH
compared with the analogues except 6a and 6a in Table 1. Among them, 6d and 6j exhibited activities
5
9
with IC50 of about 51 µM, and 6c, 6e, 6f, 6g with IC50s below 20 µM. The results indicated that the
5
N -chloroacetyl group in series 2 could increase the inhibition against both tumor cells and MetH.
Contrasted to 6b without substituent on benzene ring, alkyl substituted compounds 6a , 6c, 6d and 6e
5
showed more potent activities. They inhibited MetH activities in the following order of potency
(
substituents on benzene moiety are noted in parentheses): 6a (5.66 µM, methyl group) > 6c (12.1 µM,
5
ethyl group) > 6e (13.5 µM, tert-butyl group) > 6d (46.9 µM, isopropyl group). 6f and 6g with halogens
substituted on the p-position of benzene maintained the potent inhibition as IC50 values 4.99 µM and 14.9
µM. But the methoxyl group, whether in o-, m-position or p-position (6j, 6i, 6h), didn’t cause any
significant increase of potency (IC values as >100 µM, >100 µM and 51.5 µM).
5
0
5
Though with chloracetyl group on N position, compounds 6b, 6h and 6i didn’t show MetH inhibition.
Their normal inhibitory activity against the five tumor cells revealed that they might act on folic acid
metabolizing enzyme such as DHFR or thymidylate synthetase based their folate-like structure of substrate
5
[
47]. Or their N -chloroacetamido group might afford certain effect similar with biological alkylating agent
[48].
9

2
.4 Synthesis and in vitro Inhibitory Activities of Series 3.
Scheme 3. Reagents and conditions: (a) CH ONa, CH OH, reflux, 8 h; (b) 0.4 MPa H2, PtO , 0.5 mol/L
3
3
2
HCl, ethanol, rt; (c) acetyl chloride, anhydrous DMF, rt; (d) saturated NH in dry methanol, rt, 12 h; (e)
3
CH ONa, CH OH, reflux, 5 h.
3
3
5
The study of structure-activity relationship revealed the importance of N -chloracetyl and the substitution
of benzene in target compounds for the activities against MetH. To test the indispensability of the amino
groups at 2 and 4 position for the inhibition of MetH activities, amino group was replaced by chloride or
methoxyl group as in series 3. The synthetic route was outlined in Scheme 3. With compound 3j in hand as
in series 2, methoxylation of 2,4-chloride group with sodium methoxide in methanol gave dimethoxylated
compound 4j in 72% yield. Selectively amination on 4-chloride group of compound 3j gave 4j in good
1
2
yield which was then methoxylated on the 2-chloride to afford 4j in 70% yield. Compounds 5j , 5j and
3
1
2
5
j were then prepared by hydrogenation of the carbon-carbon double bonds and pyridine ring of 4j , 4j
3 1 2
5
and 4j with H and PtO in excellent yield, followed by N -chloroacetylation to get 6j , 6j and 6j .
3
2
2
1
2
3
The IC s of 6j , 6j and 6j against MetH were all above 100 µM, which proved that 2,4-diamino of
50
1
2
3
5
N -chloracetyl derivatives was the necessary group for inhibition of MetH. The further research was in the
molecular modeling section. It is noticed that compounds 6j -6j exhibited good inhibition effect against
1
3
5
H1299, A549, HL-60, HeLa and HT29 tumor cells. So the action of N -chloroacetyl group in 6j , 6j and
1
2
6
j3 as possible alkylation agent or on folate metabolic enzymes besides MetH will be explored in the future
[47, 48].
1
0

Table 3 Chemical structures of 6j -6j and in vitro inhibitory activities against tumor cells and MetH
1
3
b
Tumor cells (IC , µM)
50
1
2
c
Compounds
R
R
MetH
H1299
A549
>10
HL-60
HeLa
HT29
6
6
6
j1
j2
j3
OCH
3
3
OCH
3
0.47±0.03
0.32±0.07
0.41±0.08
3.41±0.16
0.24±0.02
6.80±0.50
0.94±0.19
1.83±0.48
4.54±0.30
2.52±0.21
4.82±0.70
5.69%
45.6%
31.5%
Cl
NH
NH
3.35±0.41
2
a
OCH
ND
0.51±0.02
2
a
b
c
Not tested. Indicates the average value of three independent experiments. Inhibition% at 100 µM.
2
.5 Action Mechanism of the MetH Inhibition.
We next studied the preliminary mechanism of action of the MetH inhibition by compound 6c via three
different concentrations of the substrate MTHF at a fixed concentration of Hcy (500 µM) and cell lysates.
As illustrated in Figure 5, inhibition of MetH by 6c decreased with increasing concentrations of MTHF.
When addition of MTHF at 63 µM, IC50 value of 6c was 4.46 µM. And when addition with higher
concentration of MTHF at 126 and 252 µM, lower inhibition of 6c was detected with IC50 values as 7.64
and 12.1 µM, respectively. At high substrate concentrations the action of 6c was overcome which indicated
that compound 6c was a competitive inhibitor to MetH.
1
1
20
00
63 µM
1
2
26 µM
52 µM
80
60
40
20
0
0
5
10
15
20
25
30
Compound 6c/µM
1
1

Figure 5. Effect of compound 6c on MetH activity of HL-60 at MTHF concentrations of 63, 126 and 252
µM.
2
.6 Dose-response Curves of 6c and 6a5.
Cell proliferation inhibition of compounds 6c and 6a was measured over 0.3125, 0.625, 1.25, 2.5, 5 and
5
1
0 µM in 24 h, 48 h and 72 h. When at 0.3125 and 0.625 µM, 6c caused growth inhibition of cancer cells
below 50% even after 72 h. When 6c was 1.25 µM, the inhibition was below 50% after 48 h, and about 95%
after 72 h. When 6c was 2.5 µM and above, the inhibition was about 95% even after 48 h. Compound 6a5
of 1.25 µM exhibited the inhibition below 50% after 48 h and about 65% after 72 h. Compound 6a of 1. 25,
5
2
.5, 5 and 10 µM did not show the activities as good as 6c, but the effect of concentration and time were
consistent with what had been observed in compound 6c. They exhibited a dose-response curve and a
time-response curve.
Figure 6 Dose-dependent effects of 6c were incubated for 24, 48 and 72 h. A549 cell viabilities (densities)
were determined by MTS assay. Each data point represents the average value from three independent
experiments.
2
.7 Cell cycle assay of 6c on HL-60 cells.
The effects of 6c on the cell-cycle progression in HL-60 cells were investigated as well. After 24 h
treatment with HL-60 cells, 0.5 µM and 0.75 µM of 6c was found to significantly arrest cell cycle
progression at G cells in a dose-dependent manner (36.99 and 61.97 versus 32.08% in control) (Figure 7) .
1
And a decrease in the amount of G /M cells (5.69, and 0.34 versus 11.21% in control) was observed too.
2
The results suggested that compound 6c induced a G cell cycle block, which could slow down the
1
1
2

proliferation of HL-60 cells. The data of the cell cycle assay indicated that compound 6c inhibited HL-60
proliferation in the same way as the classic inhibitors of MetH [29, 49].
A
B
C
control
0.5 µM
0.75 µM
Cell cycle distribution
D
G2
S
G1
0
5
.34
.69
0
.75 µM
37.69
61.97
0.5 µM
57.33
3
6.99
1
1.21
DMSO
56.72
3
2.08
0
10
20
30
40
50
60
70
80
DMSO
32.08%
56.72%
11.21%
0.5 µM
36.99%
57.33%
5.69%
0.75 µM
61.97%
37.69%
0.34%
G1
S
G2
Figure 7. Cell cycle assay. Cell cycle distribution was detected by flow cytometry. A) DMSO; B)
Compound 6c (0.5 µM); C) Compound 6c (0.75 µM); D) The percentage of cells in different phase of cell
cycle.
2
.8 Apoptosis analysis.
HL-60 cells were treated with compound 6c (0.5 µM and 0.75 µM) for 48 h to assess its effect on
induction of apoptosis. The fraction of cells in late apoptosis was 2.35%, 2.18%, and 70.04% for control
cells, cells treated with compound 6c (0.5 µM), and cells treated with compound 6c (0.75 µM), respectively
(Figure 8). The corresponding fractions of cells in early apoptosis were 2.32%, 3.97%, and 8.71%. These
results indicated that higher concentrations of compound 6c induced more apoptosis, especially in the in
1
3

late apoptosis which was 30-fold after being exposed to 0.75 µM of 6c. The data suggested that 6c blocked
HL-60 cells at of G phase and then induced the late apoptosis which was about the anti-proliferative effect.
1
C
A
B
0.5 µM
0.75 µM
control
D 100
UL
UR
LL
LR
9
0
0
0
0
0
0
0
0
0
0
8
7
6
5
4
3
2
1
DMSO
0.5 µM
0.75 µM
Compound Concentration (µM) UL
DMSO
UR
LL
LR
0.53 2.35 94.81 2.32
0.67 2.18 93.17 3.97
0.49 70.04 20.76 8.71
6
6
c
c
0.5
0.75
Figure 8. Analysis of apoptosis by Annexin V-FITC/PI staining and flow cytometry is shown for HL-60
cells treated with compound 6c along with untreated control. A) DMSO; B) Compound 6c (0.5 µM); C)
Compound 6c (0.75 µM); D) UL: dead cells; UR: late apoptosis cells; LL: viable cells; LR: early apoptosis
cells.
2
.9 Molecular Modeling.
In order to investigate the binding mode of the inhibitors to MetH, compound 6c was selected for
molecular docking experiments as it showed good in vitro inhibitory activity against MetH and tumor cells.
It was performed using MTHF-binding pocket of a homology model of the human MetH with the ligand
1
4

minimization of Discovery Studio 2.5. As shown in Figure 9. A, compound 6c (black) bound to MetH in
the same pocket and in same extended mode as MTHF (blue), except the difference of molecular length.
This results was consistent with our biological assay of compound 6c, which acted as MetH competitive
inhibitor. Besides the hydrogen bonds formed by 2-NH with residues Asp 525 and Asn 527, the
2
5
N -carbonyl oxygen could afford another one with residue Asn 367. This modeling result revealed that the
5
5
N -chloracetyl on the N -position was conducive to binding position and binding mode too, which was
important for the potency of MetH inhibition.
5
Compounds 6c, 6j and 6a , all with N -chloracetyl group, differ only in the substituent of benzene
5
(
p-methyl in 6c, o-methoxyl group in 6j and p-ehthyl group in 6a ). 6c (black, Figure 9. B) and 6a (yellow,
5
5
Figure 9. D) were able to establish same hydrogen bonds with the side chain of Asp-525, Asn-527 and
Asn-367. Unlike the binding mode of compounds 6c and 6a , backbone of 6j (deep red, Figure 9. C)
5
rotated and formed hydrogen bonds in different orientation because of the o-methoxyl group, which was
consistent with the reduced activity. More details were provided in the Supplementary Material (Figure 10).
5
Compared with 6c and 6j respectively, 6a (green, Figure 9. E) with chloroethyl on the N -position and 6j
6
2
(red, Figure 9. F) with chloride substitution in 2-position could establish only one hydrogen bond
interaction each with backbone residue. Accordingly IC50s of 6a and 6j against MetH were over 100 µM.
6
2
The observation could explain the reason for higher inhibition on MetH of 6c and the significant activity
5
difference between compounds with various substituents on N -position, 2,4-position and benzene ring.
1
5

Figure 9. The homology model of the human MetH based on the crystal structure of Thermotoga maritime
PDB: 1Q8J) A) Overlay of the docked pose of compound 6c (black) with MTHF (blue) in the homology
(
model of the human MetH active site. B) Docked pose of compound 6c (black). C) Docked pose of
compound 6J (deep red). D) Docked pose of compound 6a (yellow). E) Docked pose of compound 6a
5
6
(
green). F) Docked pose of compound 6J (red).
2
3
CONCLUSION
MetH inhibitors represent a kind of new and promising chemotherapeutic agents. Based on the action
5
mechanism of MetH and the previously reported inhibitors of the N -substituted tetrahydrofolate analogs, a
5
novel series of N -substituted-6-substituted pyrido[3,2-d]pyrimidine antifolates as nonclassic antifolates
were designed and tested for MetH inhibitory and antitumor activity. The structure-activity relationship for
5
these compounds was summarized. We found that N -chloracetyl substitution compounds of substituted
tetrahydrofolate analogs showed higher in vitro antiproliferative activity than others toward five cell lines
including HL-60, HeLa, A549, H1299 and HT29 and compounds 6a , 6f, and 6c showed the highest
5
activity against MetH (IC : 5.66 µM, 4.99 µM and 12.1 µM, respectively). The cell cycle distribution
5
0
assay displayed that compound 6c could increase the accumulation of G -phase cells. And compound 6c
1
showed highly potency in induction of apoptosis. Molecular modeling indicated that carbonyl of
chloracetyl group played an important role in inhibition of MetH, which could interact with amino-acid
residue Asn 367. This study revealed a new generation of lead compounds that could help in the design of
1
6

clinically useful MetH inhibitors with potential anticancer activity.
As the substrates for folate-dependent enzymes, including DHFR, TS, and MetH, shared a similar
scaffold, antifolates always exhibited multi-targeted effects currently. And a sub-structure of this series
were evaluated and revealed several compound series that possess the 2,4-diaminopyrido[3,2-d]pyrimidine
substructure that inhibit, with varying potencies, DHFR. As a consequence, off-target activity, particularly
those associated with antifolate, will be monitored in the future development of this compound class. It is
foreseen that with further optimization, the MetH inhibitors will become novel antifolates to treat cancer.
4
EXPERIMENTAL SECTION
4
.1. Chemistry
Reagents and solvents were purchased from common commercial suppliers and were used without
further purification. The melting points of the synthesized compounds were determined with a SGW® X4
1
13
apparatus. H NMR spectra were recorded at 400 MHz, and C NMR were recorded at 100 MHz on a
Bruker NMR spectrometer instrument. Tetramethylsilane was used as an internal standard to express the
chemical shift in ppm: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; and bs, broad
singlet. Mass spectra were recorded on Waters Xevo G2 Q-TOF mass spectrometer or Quattro Micro 2000.
All target compounds were assessed for purity by Agilent Technologies 1260 Infinity HPLC system with
an Extend-C18 column (4.6 mm × 150 mm, 3.5 µM). HPLC condition: methanol (CH OH): water (H O)
3
2
o
=
70: 30; flow rate, 1.0 mL/min; UV detection, from 210 to 280 nm; temperature, 25 C; injection volume,
0 µL. The purities of target compounds were confirmed to be ≥95%. Thin-layer chromatography (TLC)
was performed on silica-gel F254 plates. Compounds 4a-4j were synthesized as previously reported [44,
2
4
5].
4
.1.1 General procedure for the synthesis of intermediates 5a-5j.
A solution of 4a (100 mg, 0.36 mmol) and PtO (10%) in ethanol (30 mL) was added to 1 mL 0.5 mol/L
2
HCl in an autoclave. The autoclave was evacuated and backfilled with H three times, pressurized to 0.4
2
Mpa, and stirred at room temperature for 12 h. The reaction mixture was filtered and the filtrate was
adjusted to pH 7-8 with a saturated NaHCO solution, and evaporated to dryness under reduced pressure.
3
The resulting residue was purified by using a silica gel column (eluent: CH Cl / CH OH, 9/1) to obtain 5a
2
2
3
as a white solid.
.1.1.1 6-(4-methylphenethyl)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (5a).
4
1
7

1
Yield 92%, mp: 211-213 °C; H NMR (400 MHz, DMSO-d ) δ: 7.10 (q, J = 7.9 Hz, 4H), 6.06 (s, 2H),
6
5
1
1
1
2
4
.20 (s, 2H), 3.69 (s, 1H), 2.95 (d, J = 5.7 Hz, 1H), 2.86-2.58 (m, 2H), 2.51-2.30 (m, 2H), 2.26 (s, 3H),
1
3
.94-1.84 (m, 1H), 1.76-1.61 (m, 2H), 1.49-1.45 (m, 1H); C NMR (100 MHz, DMSO-d ) δ: 156.14,
6
55.53, 149.47, 145.63, 144.81, 139.57, 135.49, 134.92, 129.44, 129.31, 128.62, 122.84, 116.07, 111.94,
+
10.00, 106.68, 51.43, 37.89, 31.41, 28.45, 28.19, 21.08; ESI-TOF-MS: m/z calcd for C H N [(M+H) ],
1
6
22
5
84.18; found, 284.23.
.1.1.2 6-phenethyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (5b).
1
Yield 92%, mp: 183-184 °C; H NMR (400 MHz, DMSO-d ) δ: 7.50-7.00 (m, 5H), 6.00 (s, 2H), 5.16 (s,
6
2
1
1
H), 3.69 (s, 1H), 2.97 (d, J = 6.3 Hz, 1H), 2.87-2.63 (m, 2H), 2.50-2.27 (m, 2H), 2.00-1.86 (m, 1H),
13
.82-1.63 (m, 2H), 1.52-1.45 (m, 1H); C NMR (100 MHz, DMSO-d ) δ: 156.20, 155.52, 145.85, 142.75,
6
+
28.74, 126.10, 116.04, 51.48, 37.83, 31.87, 28.50, 28.21; HRMS (TOF ES ): m/z calcd for C H N
1
5
20
5
+
[
(M+H) ], 270.1719, found, 270.1714.
4
.1.1.3 6-(4-ethylphenethyl)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (5c).
1
Yield 90%, mp: 182-183 °C; H NMR (400 MHz, DMSO-d ) δ: 7.13 (q, J = 8.2 Hz, 4H), 6.19 (s, 2H),
6
5
.32 (s, 2H), 3.76 (s, 1H), 2.96 (d, J = 6.9 Hz, 1H), 2.80-2.64 (m, 2H), 2.56 (q, J = 7.6 Hz, 2H), 2.48-2.32
1
3
(
m, 2H), 1.94-1.85 (m, 1H), 1.84 -1.62 (m, 2H), 1.59-1.38 (m, 1H), 1.16 (t, J = 7.6 Hz, 3H); C NMR (100
MHz, DMSO-d ) δ: 155.81, 155.71, 144.65, 141.38, 139.84, 128.67, 128.11, 116.12, 51.38, 37.84, 31.42,
6
2
8.24, 28.11, 28.06, 16.18.
4
.1.1.4 6-(4-isopropylphenethyl)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (5d).
1
Yield 94%, mp: 186-188 °C; H NMR (400 MHz, CDCl ) δ: 7.26 -7.06 (m, 4H), 4.85 (s, 2H), 4.53 (s,
3
2
1
1
2
4
H), 3.07 (d, J = 6.1 Hz, 1H), 2.99-2.84 (m, 1H), 2.77 (t, J = 7.2 Hz, 2H), 2.64 (s, 2H), 2.16-2.02 (m, 2H),
1
3
.85 (d, J = 6.7 Hz, 2H), 1.65-1.50 (m, 1H), 1.25 (d, J = 6.7 Hz, 6H); C NMR (100 MHz, CDCl ) δ:
3
57.13, 156.86, 151.04, 146.56, 139.19, 128.32, 126.51, 115.95, 52.24, 37.46, 33.70, 32.16, 28.89, 28.54,
+
4.07; ESI-TOF-MS: m/z calcd for C H N [(M+H) ], 312.22; found, 312.27.
1
8
26
5
.1.1.5 6-(4-(tert-butyl)phenethyl)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (5e).
1
Yield 93%, mp: 247-249 °C; H NMR (400 MHz, DMSO-d ) δ: 7.30 (d, J = 8.3 Hz, 2H), 7.16 (d, J = 8.2
6
Hz, 2H), 6.53 (s, 2H), 5.63 (s, 2H), 3.92 (s, 1H), 2.98 (d, J = 6.9 Hz, 1H), 2.90-2.62 (m, 2H), 2.59-2.35 (m,
1
3
2
H), 2.47-2.36 (m, 1H), 2.01-1.84 (m, 1H), 1.85-1.61 (m, 2H), 1.59-1.39 (m, 1H), 1.26 (s, 9H); C NMR
1
8

(
100 MHz, DMSO-d ) δ: 156.24, 155.14, 148.34, 144.71, 139.52, 128.41, 125.45, 116.25, 51.24, 37.63,
6
3
4.51, 31.69, 31.24, 27.66, 27.11.
4
.1.1.6 6-(4-fluorophenethyl)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (5f).
1
Yield 90%, mp: 215-217 °C; H NMR (400 MHz, DMSO-d ) δ 7.27 (dd, J = 8.1, 5.9 Hz, 2H), 7.10 (t, J
6
=
8.8 Hz, 2H), 6.03 (s, 2H), 5.18 (s, 2H), 3.67 (s, 1H), 2.96 (d, J = 5.8 Hz, 1H), 2.86-2.63 (m, 2H),
1
3
2
1
2
4
.50-2.30 (m, 2H), 1.89 (m, 1H), 1.71 (m, 2H), 1.48 (m, 1H); C NMR (100 MHz, DMSO-d ) δ: 162.19,
6
59.80, 156.25, 155.59, 145.94, 138.82, 130.46, 130.38, 116.00, 115.47, 115.26, 51.38, 30.97, 28.49,
8.22.
.1.1.7 6-(4-chlorophenethyl)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (5g).
1
Yield 94%, mp: 172-174 °C; H NMR (400 MHz, DMSO-d ) δ: 7.34 (d, J = 8 Hz, 2H), 7.27 (d, J = 8.0
6
Hz, 2H), 6.26 (s, 2H), 5.40 (s, 2H), 3.78 (s, 1H), 2.96 (d, J = 5.9 Hz, 1H), 2.87-2.66 (m, 2H), 2.51-2.32 (m,
1
3
2
H), 1.89 (d, J = 12.4 Hz, 1H), 1.81-1.63 (m, 2H), 1.48 (s, 1H); C NMR (100 MHz, DMSO-d ) δ: 155.91,
6
1
55.63, 141.76, 130.69, 130.65, 128.75, 128.66, 116.04, 51.26, 37.52, 31.08, 27.96, 27.86; HRMS (TOF
+
+
ES ): m/z calcd for C H ClN [(M+H) ], 304.1329; found, 304.1325.
15
19
5
4
.1.1.8 6-(4-methoxyphenethyl)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (5h).
1
Yield 92%, mp: 186-188 °C; H NMR (400 MHz, DMSO-d ) δ: 7.15 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6
6
Hz, 2H), 6.66 (s, 2H), 6.01 (s, 2H), 3.72 (s, 3H), 3.02-2.92 (m, 1H), 2.83-2.57 (m, 2H), 2.50-2.37 (m, 1H),
1
3
1
.87 (s, 3H), 1.80-1.55 (m, 2H), 1.54-1.35 (m, 1H); C NMR (100 MHz, DMSO-d ) δ: 157.82, 156.53,
6
1
54.59, 140.64, 134.44, 129.64, 116.20, 114.18, 55.42, 51.18, 37.81, 30.85, 27.50, 26.60; ESI-TOF-MS:
+
m/z calcd for C H N O [(M+H) ], 300.18; found, 300.19.
1
6
22
5
4
.1.1.9 6-(3-methoxyphenethyl)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (5i).
1
Yield 90%, mp: 196-198 °C; H NMR (400 MHz, DMSO-d ) δ: 7.19 (t, J = 8.0 Hz, 1H), 6.81 (d, J = 6.3
6
Hz, 2H), 6.74 (d, J = 8.4 Hz, 1H), 5.97 (s, 2H), 5.12 (s, 2H), 3.73 (s, 3H), 3.65 (s, 1H), 2.96 (d, J = 5.4 Hz,
1
H), 2.84-2.63 (m, 2H), 2.49-2.31 (m, 2H), 1.90 (d, J = 12.8 Hz, 1H), 1.83-1.61 (m, 2H), 1.61-1.42 (m, 1H);
1
3
C NMR (100 MHz, DMSO-d ) δ: 159.76, 156.38, 155.44, 146.36, 144.38, 129.72, 121.01, 116.02, 114.43,
6
+
+
1
3
4
11.51, 55.34, 51.51, 37.71, 31.93, 28.68, 28.29; HRMS (TOF ES ): m/z calcd for C H N O [(M+H) ],
16 22 5
00.1824; found, 300.1820.
.1.1.10 6-(2-methoxyphenethyl)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (5j)
1
Yield 92%, mp: 189-191 °C; H NMR (400 MHz, DMSO-d ) δ: 7.17 (d, J = 5.7 Hz, 2H), 6.94 (d, J = 8.4
6
1
9

Hz, 1H), 6.87 (t, J = 7.3 Hz, 1H), 6.29 (s, 2H), 5.72 (s, 2H), 4.21 (s, 1H), 3.78 (s, 3H), 2.96 (d, J = 7.1 Hz,
13
1
H), 2.80-2.60 (m, 2H), 2.50-2.31 (m, 2H), 1.98-1.89 (m, 1H), 1.79-1.56 (m, 2H), 1.55-1.40 (s, 1H); C
NMR (100 MHz, DMSO) δ 157.51, 130.39, 129.91, 127.55, 120.71, 116.11, 111.03, 63.40, 55.66, 51.53,
+
3
6.00, 27.76, 27.51, 26.39; ESI-TOF-MS: m/z calcd for C H N O [(M+H) ], 300.18; found, 300.29.
1
6
22
5
4
.1.2 General procedure for the synthesis of title compounds 6a , 6b-6j & 6j -6j .
5 1 3
A solution of 5a (100 mg, 0.35 mmol) in anhydrous DMF (3 mL) in an ice bath for 30 min was added to
chloroacetyl chloride (100 µL, 1.26 mmol) and the reaction mixture was stirred for about 30 min. Then the
mixture was extracted with CH Cl . The organic layers were combined, washed with saturated NaHCO
3
2
2
and water, dried over anhydrous Na SO , filtered, and concentrated. The residue was purified by column
2
4
chromatography over silica gel, eluting with CH Cl /CH OH (30/1), to give 6a as a white solid.
2
2
3
5
4
.1.2.1 6-(4-methylphenethyl)-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (6a5).
1
Yield 64%, mp: 159-160 °C; H NMR (400 MHz, DMSO-d ) δ 7.06 (s, 4H), 6.56 (s, 2H), 6.05 (s, 2H),
6
4
.87-4.54 (m, 1H), 4.33-3.94 (m, 2H), 2.71-2.45 (m, 2H), 2.44-2.11 (m, 6H), 1.52-1.43 (m, 2H), 1.38-1.21
13
(
m, 1H); C NMR (100 MHz, DMSO-d ) δ 167.04, 164.98, 161.54, 159.19, 139.40, 129.25, 128.64,
6
+
1
05.60, 52.31, 43.58, 36.95, 31.82, 29.58, 28.31, 21.08; HRMS (TOF ES ): m/z calcd for C H ClN O
18
23
5
+
[
(M+H) ], 360.1591; found, 360.1582.
4
.1.2.2 6-phenethyl-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (6b).
1
Yield 63%, mp: 166-168 °C; H NMR (400 MHz, DMSO-d ) δ: 7.25 (t, J = 7.3 Hz, 2H), 7.21-7.12 (m,
6
3
H), 6.57 (s, 2H), 6.05 (s, 2H), 4.87-4.63 (m, 1H), 4.23-4.11 (m, 2H), 2.72-2.54 (m, 2H), 2.28-2.23 (m, 3H),
13
1
.65-1.45 (m, 2H), 1.34-1.28 (m, 1H); C NMR (100 MHz, DMSO-d ) δ: 167.07, 165.64, 164.98, 162.63,
6
1
61.54, 160.81, 159.20, 150.86, 142.56, 140.48, 128.76, 128.68, 128.14, 126.11, 124.16, 105.59, 52.35,
+
+
4
3.59, 36.89, 32.28, 29.60, 28.31; HRMS (TOF ES ): m/z calcd for C H ClN O [(M+H) ], 346.1435;
17 21 5
found, 346.1435.
4
.1.2.3 6-(4-ethylphenethyl)-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (6c).
1
Yield 65%, mp: 137-138 °C; H NMR (400 MHz, DMSO-d ) δ: 7.08 (s, 4H), 6.57 (s, 2H), 6.06 (s, 2H),
6
4
.80-4.65 (m, 1H), 4.21-4.13 (m, 2H), 3.39-3.34 (m, 1H), 2.72-2.51 (m, 4H), 2.35-2.15 (m, 2H), 1.57-1.23
1
3
(
m, 3H), 1.16-1.09 (m, 3H); C NMR (100 MHz, DMSO-d ) δ: 167.06, 164.92, 161.52, 159.21, 141.40,
6
+
1
39.69, 128.68, 128.06, 105.61, 65.39, 52.32, 43.59, 36.92, 31.85, 29.57, 28.23, 16.18; HRMS (TOF ES ):
+
m/z calcd for C H ClN O [(M+H) ], 374.1748; found, 374.1745.
1
9
25
5
2
0

4
.1.2.4
6-(4-isopropylphenethyl)-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine
(6d).
1
Yield 63%, mp: 152-154 °C; H NMR (400 MHz, DMSO-d ) δ: 7.10 (s, 4H), 6.56 (s, 2H), 6.05 (s, 2H),
6
4
.72 (s, 1H), 4.33-4.02 (m, 2H), 2.95-2.70 (m, 1H), 2.59 (s, 2H), 2.26 (s, 3H), 1.64-1.24 (m, 3H), 1.17 (d, J
1
3
=
6.4 Hz, 6H); C NMR (101 MHz, DMSO-d ) δ: 167.06, 161.19, 159.22, 145.96, 140.18, 128.65, 126.56,
6
+
5
2.34, 43.59, 36.88, 33.49, 31.83, 29.54, 28.00, 24.44; HRMS (TOF ES ): m/z calcd for C H ClN O
20
27
5
+
[
(M+H) ], 388.1904; found, 388.1900.
4
.1.2.5
6-(4-(tert-butyl)phenethyl)-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine
(6e).
1
Yield 63%, mp: 151-153 °C; H NMR (400 MHz, DMSO-d ) δ: 7.26 (d, J = 7.7 Hz, 2H), 7.10 (d, J = 7.9
6
Hz, 2H), 6.56 (s, 2H), 6.05 (s, 2H), 4.85-4.60 (m, 1H), 4.25-4.06 (m, 2H), 2.72-2.52 (m, 2H), 2.40-2.13 (m,
1
3
3
H), 1.67-1.28 (m, 3H), 1.25 (s, 9H); C NMR (100 MHz, DMSO-d ) δ: 167.07, 164.88, 161.50, 159.22,
6
+
1
48.32, 139.43, 128.39, 125.39, 105.61, 52.32, 43.59, 36.82, 34.49, 31.68, 29.55, 28.24; HRMS (TOF ES ):
+
m/z calcd for C H ClN O [(M+H) ], 402.2061; found, 402.2058.
2
1
29
5
4
.1.2.6 6-(4-fluorophenethyl)-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (6f).
1
Yield 64%, mp: 181-183 °C; H NMR (400 MHz, DMSO-d ) δ: 7.38-7.14 (m, 2H), 7.07 (t, J = 8.6 Hz,
6
2
1
1
H), 6.58 (s, 2H), 6.06 (s, 2H), 4.85-4.58 (m, 1H), 4.28-3.98 (m, 2H), 2.74-2.51 (m, 2H), 2.35-2.16 (m, 3H),
13
.52-1.43 (m, 2H), 1.41-1.21 (m, 1H); C NMR (100 MHz, DMSO-d ) δ: 167.09, 165.00, 161.53, 159.19,
6
38.63, 130.52, 130.44, 115.42, 115.21, 105.55, 52.23, 43.59, 36.96, 31.35, 29.61, 28.31; HRMS (TOF
+
+
ES ): m/z calcd for C H ClFN O [(M+H) ], 364.1340; found, 364.1331.
17
20
5
4
.1.2.7 6-(4-chlorophenethyl)-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (6g).
1
Yield 62%, mp: 172-174 °C; H NMR (400 MHz, DMSO-d ) δ: 7.31 (d, J = 7.9 Hz, 2H), 7.22 (d, J = 8.1
6
Hz, 2H), 6.57 (s, 2H), 6.05 (s, 2H), 4.83-4.65 (m, 1H), 4.25-4.10 (m, 2H), 2.75-2.54 (m, 2H), 2.26 (s, 3H),
1
3
1
1
.56-1.41 (m, 2H), 1.36-1.25 (s, 1H); C NMR (100 MHz, DMSO-d ) δ: 165.30, 161.56, 160.49, 159.18,
6
+
51.45, 141.58, 130.68, 128.60, 105.53, 52.22, 43.59, 36.68, 31.48, 29.62, 28.31; HRMS (TOF ES ): m/z
+
calcd for C H Cl N O [(M+H) ], 380.1045; found, 380.1039.
17
20
2
5
4
.1.2.8 6-(4-methoxyphenethyl)-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (6h).
1
Yield 65%, mp: 144-145 °C; H NMR (400 MHz, DMSO-d ) δ: 7.09 (d, J = 7.8 Hz, 2H), 6.81 (d, J = 7.9
6
Hz, 2H), 6.66 (s, 2H), 6.14 (s, 2H), 4.90-4.50 (m, 1H), 4.32-3.96 (m, 2H), 3.70 (s, 3H), 2.80-2.41 (m, 3H),
2
1

13
2
.40-2.09 (m, 2H), 1.85-1.20 (m, 3H); C NMR (100 MHz, DMSO-d ) δ: 167.07, 161.06, 159.32, 157.81,
6
+
1
34.36, 129.67, 129.59, 114.11, 105.73, 55.41, 52.25, 43.59, 36.97, 31.32, 29.44, 27.98; HRMS (TOF ES ):
+
m/z calcd for C H ClN O [(M+H) ], 376.1540; found, 376.1538.
1
8
23
5
2
4
.1.2.9 6-(3-methoxyphenethyl)-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (6i).
1
Yield 60%, mp: 161-162 °C; H NMR (400 MHz, DMSO-d ) δ: 7.16 (t, J = 7.7 Hz, 1H), 6.76-6.71 (m,
6
3
2
1
1
H), 6.58 (s, 2H), 6.06 (s, 2H), 4.87-4.62 (m, 1H), 4.21-4.13 (m, 2H), 3.72 (s, 3H), 2.70-2.52 (m, 2H),
1
3
.39-2.13 (m, 3H), 1.53-1.45 (m, 2H), 1.34-1.29 (m, 1H); C NMR (100 MHz, DMSO-d ) δ: 167.09,
6
65.01, 163.80, 161.52, 161.36, 159.70, 159.57, 159.21, 144.17, 129.66, 121.00, 114.45, 112.10, 111.58,
+
05.59, 55.33, 52.31, 43.59, 36.78, 32.31, 29.60, 28.29; HRMS (TOF ES ): m/z calcd for C H ClN O
1
8
23
5
2
+
[
(M+H) ], 376.1540; found, 376.15389
4
.1.2.10
6-(2-methoxyphenethyl)-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine
(6j).
1
Yield 59%, mp: 147-149 °C; H NMR (400 MHz, DMSO-d ) δ: 7.20-7.09 (m, 2H), 6.91 (d, J = 8.0 Hz,
6
1
2
1
2
3
4
H), 6.83 (t, J = 7.4 Hz, 1H), 6.54 (s, 2H), 6.06 (s, 2H), 4.81-4.65 (m, 1H), 4.31-4.05 (m, 2H), 3.74 (s, 3H),
1
3
.62-2.49 (m, 2H), 2.38-2.15 (m, 3H), 1.70-1.21 (m, 4H); C NMR (100 MHz, DMSO-d ) δ: 167.05,
6
64.76, 161.49, 159.23, 157.44, 130.19, 130.08, 127.59, 120.64, 111.01, 105.69, 55.63, 52.49, 43.58, 34.95,
+
+
9.48, 28.21, 26.99; HRMS (TOF ES ): m/z calcd for C H ClN O [(M+H) ], 376.1540; found,
1
8
23
5
2
76.1538.
.1.2.11 2,4-dimethoxy-6-(2-methoxyphenethyl)-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine
6j1).
Yield 82%; H NMR (400 MHz, CDCl ) δ: 7.16 (t, J = 7.6 Hz, 1H), 7.12-7.00 (m, 1H), 6.86 (t, J = 7.4
(
1
3
Hz, 1H), 6.80 (d, J = 8.1 Hz, 1H), 4.98 (s, 1H), 4.17-3.96 (m, 8H), 3.74 (s, 3H), 2.79-2.67 (m, 2H),
1
3
2
1
2
4
4
2
.66-2.52 (m, 2H), 2.47-2.30 (m, 1H), 1.82-1.47 (m, 3H); C NMR (100 MHz, CDCl ) δ: 166.82, 166.06,
3
64.42, 162.28, 157.30, 129.85, 127.23, 120.44, 112.78, 110.20, 55.12, 54.49, 52.43, 42.38, 33.31, 28.55,
+
+
7.89, 26.98, 14.43; HRMS (TOF ES ): m/z calcd for C H ClN O [(M+H) ], 406.1534; found,
2
0
25
3
4
06.1534.
.1.2.12
-chloro-4-amino-6-(2-methoxyphenethyl)-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine (6j2).
2
2

1
Yield 63%, mp: 143-145 °C; H NMR (400 MHz, CDCl ) δ: 7.26-7.03 (m, 2H), 6.96-6.79 (m, 2H),
3
5
.82-5.46 (d, 2H), 4.26-3.91 (m, 3H), 3.78 (s, 3H), 3.04-2.50 (m, 4H), 2.36 (d, J = 5.4 Hz, 1H), 2.13-1.46
1
3
(
m, 3H); C NMR (100 MHz, CDCl ) δ: 165.26, 161.64, 159.78, 157.23, 156.64, 129.84, 128.17, 127.89,
3
+
1
20.81, 115.08, 110.66, 55.32, 54.49, 41.58, 31.32, 27.17, 26.71, 26.20; HRMS (TOF ES ): m/z calcd for
+
C H Cl N O [(M+H) ], 395.1042; found, 395.1041.
1
8
21
2
4
2
4
2
.1.2.13
-methoxy-4-amino-6-(2-methoxyphenethyl)-5-chloracetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine (6j3).
1
Yield 60%, mp: 46-48 °C; H NMR (400 MHz, CDCl ) δ: 7.26-7.01 (m, 2H), 7.01-6.65 (m, 2H), 5.31 (s,
3
1
3
1
H), 5.07-4.72 (m, 1H), 4.39-3.52 (m, 7H), 2.97-2.23 (m, 5H), 2.02-1.29 (m, 4H); C NMR (100 MHz,
CDCl ) δ: 132.36, 130.93, 129.88, 128.85, 127.77, 127.44, 120.73, 110.55, 110.47, 71.81, 55.27, 54.67,
3
5
3.53, 42.44, 41.76, 34.40, 31.51, 30.71, 29.70, 29.45, 28.24, 27.72, 27.48, 26.80, 26.22; HRMS (TOF
+
+
ES ): m/z calcd for C H ClN O [(M+H) ], 391.1537; found, 391.1540.
19
24
4
3
4
.1.3 6-(4-methylphenethyl)-5-formyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (6a1).
A solution of 5a (50 mg, 0.17 mmol) in 10 mL CH Cl was added formic acid (0.5 mL) and the
2
2
mixture was at room temperature stirred for 1 h. Then acetic anhydride (0.5 mL) was added and the
resulting solution was stirred for 0.5 h. The mixture was adjusted to pH 7 with a saturated NaHCO3
solution and extracted with CH Cl . The organic layer was separated, dried over anhydrous Na SO ,
2
2
2
4
filtered, and concentrated. The residue was purified by column chromatography over silica gel, eluting with
1
CH Cl /CH OH (9/1), to give 6a as a white solid. Yield 91%, mp: 150-152 °C; H NMR (400 MHz,
2
2
3
1
DMSO-d ) δ: 8.20&8.07 (s, 1H), 7.11-6.99 (m, 4H), 6.45 (s, 1H), 6.91 (s, 3H), 4.62-4.49&4.05-3.91 (m,
6
1
3
1
H), 2.64-2.40 (m, 3H), 2.41-2.30 (m, 1H), 2.24 (s, 3H), 2.16-1.96 (m, 1H), 1.83-1.48 (m, 3H); C NMR
(
100 MHz, DMSO-d ) δ: 161.47, 161.11, 160.67, 160.53, 159.18, 158.98, 158.94, 141.57, 141.42, 139.60,
6
1
2
4
39.10, 128.72, 128.63, 128.18, 128.06, 53.66, 48.29, 34.24, 33.53, 31.74, 31.67, 28.47, 28.22, 27.87,
+
7.03, 26.51, 21.55, 16.16; ESI-TOF-MS: m/z calcd for C H N O [(M+H) ], 312.18; found, 312.28.
1
7
22
5
.1.4 6-(4-methylphenethyl)-5-acryloyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (6a2).
A solution of 5a (100 mg, 0.35 mmol) and 20 mg NaHCO in anhydrous DMF (3 mL) was added to
3
acryloyl chloride (100 µL, 1.24 mmol) and the reaction mixture was at room temperature stirred for about
0 min. Then the mixture was extracted with CH Cl . The organic layers were combined, washed with
3
2
2
saturated NaHCO and water, dried over anhydrous Na SO , filtered, and concentrated. The residue was
3
2
4
2
3

purified by column chromatography over silica gel, eluting with CH Cl /CH OH (30/1), to give 6a as a
2
2
3
2
1
white solid. Yield 58%, mp: 207-209 °C; H NMR (400 MHz, DMSO-d ) δ: 7.12-7.01 (m, 4H), 6.38 (s,
6
2
H), 6.31-6.14 (m, 2H), 6.00 (s, 2H), 5.70-5.57 (m, 1H), 5.06-4.60 (m, 1H), 2.66-2.48 (m, 2H), 2.35-2.08
1
3
(
m, 6H), 1.68-1.33 (m, 3H); C NMR (100 MHz, DMSO-d ) δ: 165.68, 163.45, 161.28, 159.69, 139.49,
6
1
34.89, 129.79, 129.22, 128.68, 127.93, 106.29, 51.36, 36.62, 31.88, 29.47, 28.09, 21.08; HRMS (TOF
+
+
ES ): m/z calcd for C H N O [(M+H) ], 338.1981; found, 338.1988.
19
24
5
4
.1.5 6-(4-methylphenethyl)-5-allyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (6a3).
A solution of 5a (100 mg, 0.35 mmol) and 111 mg K CO in anhydrous DMF (5 mL) was added to allyl
2
3
bromide (70 µL, 1.04 mmol) and the reaction mixture was at room temperature stirred for 48 h. Then the
mixture was extracted with CH Cl . The organic layers were combined, washed with water, dried over
2
2
anhydrous Na SO , filtered, and concentrated. The residue was purified by column chromatography over
2
4
o
1
silica gel, eluting with CH Cl /CH OH (30/1), to give 6a as a white solid. Yield 60%, mp: 69-71 C; H
2
2
3
3
NMR (400 MHz, DMSO-d ) δ: 7.03 (s, 4H), 6.16-5.99 (m, 1H), 5.90 (s, 2H), 5.51 (s, 2H), 5.21 (d, J = 17.1
6
Hz, 1H), 5.10 (d, J = 10.1 Hz, 1H), 3.39-3.31 (m, 1H), 3.18-3.05 (m, 1H), 2.91 (d, J = 2.9 Hz, 1H),
2
1
1
.74-2.61 (m, 1H), 2.56-2.45 (m, 1H), 2.47 (s, 1H), 2.40-2.26 (m, 2H), 2.23 (s, 3H), 2.15-1.99 (m, 1H),
1
3
.53-1.34 (m, 3H); C NMR (100 MHz, DMSO-d ) δ: 160.37, 158.76, 153.93, 139.79, 137.76, 134.77,
6
+
29.19, 128.63, 117.15, 116.20, 56.25, 52.44, 33.58, 32.18, 24.98, 21.06, 19.79; HRMS (TOF ES ): m/z
+
calcd for C H N [(M+H) ], 324.2188; found, 324.2184.
19
26
5
4
.1.6
6a4).
A solution of5a (100 mg, 0.35 mmol) and 16 mg K CO in anhydrous DMF (3 mL) was added to
6-(4-methylphenethyl)-5-cyclopropionyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine
(
2
3
isopropanecarbonyl chloride (100 µL, 1.10 mmol) and the reaction mixture was at room temperature stirred
for 30 min. Then the mixture was extracted with CH Cl . The organic layers were combined, washed with
2
2
saturated NaHCO and water, dried over anhydrous Na SO , filtered, and concentrated. The residue was
3
2
4
purified by column chromatography over silica gel, eluting with CH Cl /CH OH (30/1), to give 6a as a
2
2
3
4
1
white solid. Yield 63%, mp: 90-92 °C; H NMR (400 MHz, DMSO-d ) δ: 7.05 (s, 4H), 6.38 (s, 2H), 5.97 (s,
6
2
0
H), 4.87-4.66 (m, 1H), 2.64-2.50 (m, 2H), 2.33-2.19 (m, 5H), 2.20-2.09 (m, 1H), 1.65-1.30 (m, 4H),
13
.98-0.87 (m, 1H), 0.80-0.51 (m, 3H); C NMR (100 MHz, DMSO-d ) δ: 173.86, 163.14, 161.18, 159.99,
6
2
4

1
39.50, 134.87, 129.21, 128.66, 107.09, 51.20, 36.51, 31.99, 29.40, 28.00, 21.08, 12.44, 8.49, 8.35; HRMS
+
+
(
TOF ES ): m/z calcd for C H N O [(M+H) ], 352.2137; found, 352.2139.
20 26 5
4
.1.7
6-(4-methylphenethyl)-5-(2-chloroethyl)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine
(6a6).
A solution of 6a (50 mg, 0.14 mmol) and BH -THF (1 mL, 1 mol/L) in anhydrous THF (1 mL) was at
5
3
room temperature stirred overnight. The mixture was added 20% HCl and stirred for 1 h. Then the mixture
was adjusted to pH 7 with a saturated NaHCO solution, and evaporated to dryness under reduced pressure.
3
The residue was purified by column chromatography over silica gel, eluting with CH Cl /CH OH (30/1), to
2
2
3
1
give 6a as a white solid. Yield 75%, mp: 152-154 °C; H NMR (400 MHz, DMSO-d ) δ: 7.22-6.87 (m, 4H),
6
6
6
2
.32 (s, 2H), 5.84 (s, 2H), 4.02-3.89 (m, 1H), 3.82-3.64 (m, 1H), 3.09-2.81 (m, 3H), 2.78-2.63 (m, 1H),
1
3
.63-2.50 (m, 1H), 2.42-2.29 (m, 2H), 2.24 (s, 3H), 2.14-2.01 (m, 1H), 1.57-1.40 (m, 3H). C NMR (100
MHz, DMSO-d ) δ: 160.57, 157.94, 139.72, 134.87, 129.30, 128.51, 115.67, 54.45, 52.76, 43.79, 33.22,
6
+
+
3
3
4
2.18, 29.67, 24.24, 21.06. HRMS (TOF ES ): m/z calcd for C H ClN [(M+H) ], 346.1798; found,
18 25 5
46.1796.
.1.8 6-(4-methylphenethyl)-5-(3-chloropropionyl)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine
(
6a7).
A solution of 5a (100 mg, 0.35 mmol) in anhydrous DMF (3 mL) was added to bromoacetyl bromide
100 µL, 1.15 mmol) and the reaction mixture was at room temperature stirred for 30 min. Then the
(
mixture was extracted with CH Cl . The organic layers were combined, washed with saturated NaHCO
3
2
2
and water, dried over anhydrous Na SO , filtered, and concentrated. The residue was purified by column
2
4
chromatography over silica gel, eluting with CH Cl /CH OH (30/1), to give 6a as a white solid. Yield 51%,
2
2
3
7
1
mp: 151-153 °C; H NMR (400 MHz, CDCl ) δ: 7.20-7.01 (m, 4H), 5.20-4.90 (m, 3H), 4.73 (s, 1H),
3
1
3
3
.98-3.67 (m, 2H), 3.07-2.12 (m, 11H), 1.98-1.54 (m, 3H); C NMR (100 MHz, CDCl ) δ: 171.20, 129.40,
3
+
1
29.16, 128.24, 52.58, 40.32, 36.62, 32.58, 31.42, 29.92, 28.30, 20.98; HRMS (TOF ES ): m/z calcd for
+
C H ClN O [(M+H) ], 374.1748; found, 374.1749.
1
9
25
5
4.1.9 6-(4-methylphenethyl)-5-(3-chloropropyl)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine
(
6a8).
A procedure similar to the preparation of 6a was followed to prepare 6a from 3a . The yield was 73%
6
7
8
1
as a colorless oil; H NMR (400 MHz, DMSO-d ) δ: 7.05 (s, 4H), 6.68 (s, 2H), 6.35 (s, 2H), 3.79-3.61 (m,
6
2
5

2
H), 3.43-3.30 (m, 3H), 2.99 (s, 1H), 2.84-2.53 (m, 4H), 2.47-2.34 (s, 2H), 2.24 (s, 3H), 2.23-1.81 (m, 3H).
1
3
C NMR (100 MHz, DMSO-d ) δ: 161.47, 156.39, 156.01, 139.62, 134.86, 129.24, 128.61, 116.69, 61.19,
6
+
5
2.55, 50.67, 44.04, 33.31, 32.09, 31.33, 29.66, 21.07; HRMS (TOF ES ): m/z calcd for C H ClN
1
9
27
5
+
[
(M+H) ], 360.1955; found, 360.1961.
4
.1.10 6-(4-methylphenethyl)-5-bromoacetyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine-2,4-diamine (6a9).
A solution of 5a (100 mg, 0.35 mmol) in anhydrous DMF (3 mL) was added to bromoacetyl bromide
(100 µL, 1.15 mmol) and the reaction mixture was stirred for about 30 min. Then the mixture was extracted
with CH Cl . The organic layers were combined, washed with saturated NaHCO and water, dried over
2
2
3
anhydrous Na SO , filtered, and concentrated. The residue was purified by column chromatography over
2
4
1
silica gel, eluting with CH Cl /CH OH (30/1), to give 6a as a white solid. Yield 49%, mp: 196-198 °C; H
2
2
3
9
NMR (400 MHz, DMSO-d ) δ: 7.33-6.90 (m, 6H), 4.90-4.55 (m, 1H), 4.33-3.76 (m, 2H), 2.77-2.52 (m,
6
1
3
2
H), 2.45-2.35 (d, J = 6.5 Hz, 1H), 2.25 (s, 3H), 2.24-1.90 (m, 2H), 1.83-2.40 (m, 3H); C NMR (100
MHz, DMSO-d ) δ: 160.05, 153.57, 138.60, 135.13, 129.30, 129.25, 128.64, 128.55, 47.68, 31.95, 31.42,
6
+
+
3
0.13, 28.83, 26.42, 21.08. HRMS (TOF ES ): m/z calcd for C H BrN O [(M+H) ], 404.1086, 406.1066;
18 23 5
found, 404.1089, 406.1070.
4
.2 Evaluation of Cell Viability.
The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)
assay was used to determine the in vitro antiproliferative effect of the compounds. HL-60, HeLa, A549,
3
3
H1299 and HT29 cells were cultured in 96-well plates at an initial density of 1×10 cells/well, 1×10
3
3
3
cells/well, 2×10 cells/well, 1×10 cells/well and 3×10 cells/well in RPMI-1640 supplemented with 10%
o
fetal bovine serum, penicillinestreptomycin solution, at 37 C with 5% CO . Cells were grown for 12 h and
2
then treated with compounds at concentrations ranging from 0.0078 µM to 100 µM and incubated for 24,
4
8 or 72 h in 200 µL media. MTS reagent (20 µL) in phosphate buffer solution was added to each well and
incubated further for 3-4 h. A490 nm was measured using a 96-well plate spectrophotometer (BioRad
Benchmark plus). Three individual experiments were performed to obtain mean cell viability. MTX was
used as positive controls.
4
.3 MetH activity assay.
HL-60 cells at the density of 1×10 cells/well (900 µL) in 24-well plates were cultured overnight before
treating with various concentrations of test compounds (100 µL) for 3 h. Then the cells were harvested
6
2
6

(1,000 rmp, 10 min) and lysed with lysis buffer (150 µL). After centrifugation (10,000 rpm, 10 min, at 4
o
C), the supernatants were assayed in EP tubes for MetH activity. The assay solution contained 93 µL
deionized water, 20 µL 1 mol/L PBS (pH 7.2), 5 µL 1 mol/L DTT, 5 µL 0.76 mmol/L SAM, 5 µL 20
mmol/L Hcy, 20 µL 0.5 mmol/L OH-Cbl. After adding 40 µL cell lysates into the mixture, it was
o
preincubated at 37 C for 5 min immediately. Then the reaction was initiated by mixing with 12 µL 4.2
o
mmol/L MTHF and incubated 37 C for 10 min. The reaction was terminated by the addition of 50 µL stop
o
buffer (5 mmol/L HCl+51% formic acid) and incubated at 80 C for 10 min. After cooling, 200 µl mixture
was transferred to 96-well microplates. Then the plate was read at 350 nm by using a 96-well plate
spectrophotometer (BioRad Benchmark plus).
4
.4 Cell Cycle Assay.
HL-60 cells at a density of 0.8×10 cells per well were cultured in 60 mm petri dish overnight, then they
6
were harvested and washed twice with ice-cold PBS after treating with compound 6c (0.5 µM and 0.75 µM)
or 0.1% DMSO for 24 h. Then, these cells were fixed in 70% cooled ethanol at 4 ºC overnight, until being
washing with PBS just before analysis by flow cytometry. 0.5 mL PBS within 10 mg/mL RNase A was
o
added to the fixed cells and incubated at 37 C for 30 min in the dark. After this, 10 µL propidium iodide
(
PI) was added to the cells. The DNA contents were determined by a flow cytometry using the BD
FACSCalibur flow cytometer.
.5 Apoptosis Analysis.
HL-60 cells at a density of 0.5×10 cells per well were cultured in 60 mm petri dish overnight, then they
4
6
were harvested and washed twice with ice-cold PBS after treating with compound 6c (0.5 µM and 0.75 µM)
or 0.1% DMSO for 48 h. 400 µL BindingBuffer with 5 µL AnnexinV-FITC was add to the cells and
incubated for 15 min at room temperature in the dark. After this, 10 µL PI was added to the cells and
incubated for 5 min. Cells were assayed for apoptosis by flow cytometry using the BD FACSCalibur flow
cytometer.
4
.6 Molecular Modeling Computational Studies.
Multiple sequences of human, rat, and Thermotoga maritime methionine synthases (MS) were used in
the alignment process. The multiple sequence alignment obtained in the previous step was used together
with the crystal structure of Thermotoga maritime MS (PDB: 1Q8J) as an input for the building of the
homology model of the human MetH using Accelrys Discovery Studio client 2.5 (DS 2.5) software. The
2
7

protonation state of the proteins and the ligands were calculated using the default settings. Water molecules
in the active site were removed. The active site was defined by a sphere of 10.0 Å from the native ligand in
the homology model of the human MetH. The CDocker protocol was used to score the docked poses.
SUPPORTING INFORMATION
The Supporting Information is available free of charge on the ACS Publications Web site at The Supporting
Information is available free of charge on the ACS Publications website at DOI:
1
13
Synthesis and characterization of 5j -5j . Copies of HRMS, H NMR and C NMR spectra of final
1
3
compounds (PDF). Docking. HPLC traces of key target compound 6c.
AUTHOR INFORMATION
Corresponding Author
⊥
J.Y.L. and Z.L.Z. E-mail: jyliu@bjmu.edu.cn and lilybmu@bjmu.edu.cn
Author Contributions
*
M.W. and C.T. contributed equally to this work and are considered as co-first authors.
ACKNOWLEDGMENTS
The authors thank the Peking University Medical and Health Analysis Center for their help on the cell cycle
assay and apoptosis analysis. This work was supported by the National Natural Science Foundation of
China (21172014).
ABBREVIATIONS USED
DHFR, dihydrofolate reductase; TS, thymidylate systhase; THF, tetrahydrofuran; DMF,
N,N-dimethylformamide; DMAC, dimethylacetamide; rt, room temperature; MTX, methotrexate; PBS,
phosphate buffer saline; DTT, Dithiothreitol; SAM, s-adenosyl methionine; Hcy, homocysteine; OH-Cbl,
hydroxocobalamin; PDB, protein data bank
REFERENCES
[1] J. Hu, B. Wang, N. R. Sahyoun, Application of the key events dose-response framework to folate
metabolism, Crit. Rev. Food Sci. 56 (2016) 1325-1333.
[2] J. Stubbe, Binding site revealed of nature's most beautiful cofactor, Science 266 (1994) 1663-1664.
[
3] R. Banerjee, S. W. Ragsdale, The Many faces of vitamin B : catalysis by cobalamin-dependent
1
2
enzymes, Annu. Rev. Biochem. 72 (2003) 209-247.
[4] R. G. Matthews, Cobalamin-dependent methyltransferases, Acc. Chem. Res. 34 (2001) 681-689.
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